Optimization of Class I Histone Deacetylase PROTACs Reveals that HDAC1/2 Degradation is Critical to Induce Apoptosis and Cell Arrest in Cancer Cells

Class I histone deacetylase (HDAC) enzymes 1, 2, and 3 organize chromatin as the catalytic subunits within seven distinct multiprotein corepressor complexes and are established drug targets. We report optimization studies of benzamide-based Von Hippel–Lindau (VHL) E3-ligase proteolysis targeting chimeras (PROTACs) and for the first time describe transcriptome perturbations resulting from these degraders. By modifying the linker and VHL ligand, we identified PROTACs 7, 9, and 22 with submicromolar DC50 values for HDAC1 and/or HDAC3 in HCT116 cells. A hook effect was observed for HDAC3 that could be negated by modifying the position of attachment of the VHL ligand to the linker. The more potent HDAC1/2 degraders correlated with greater total differentially expressed genes and enhanced apoptosis in HCT116 cells. We demonstrate that HDAC1/2 degradation by PROTACs correlates with enhanced global gene expression and apoptosis, important for the development of more efficacious HDAC therapeutics with reduced side effects.


■ INTRODUCTION
Class I histone deacetylase (HDAC) enzymes, HDAC1, 2, 3, and 8, are four out of eleven zinc-dependent HDAC enzymes, catalyzing the hydrolysis of acetyl groups in N-ε-acetyl-L-lysine residues in histones and nonhistone proteins. 1 HDAC1/2 shares over 80% sequence homology, is localized in the nucleus, and exists in several multiprotein corepressor complexes including Sin3, CoREST, MiDAC, and NuRD. 1,2 HDAC3 shares approximately 50% sequence homology with HDAC1/2, is also predominantly localized in the nucleus, and exists exclusively in the SMRT/NCoR corepressor complex. 1,3 HDAC8, in contrast to HDAC1/2 and 3, can be found in both the nucleus and cytoplasm and is not present in corepressor complexes. 1,4 Four HDAC inhibitors (HDACi) have been approved by the US FDA including the hydroxamic acids vorinostat, panobinostat, and belinostat and the cyclic peptide natural product romidepsin. These drugs are primarily used for the treatment of hematological cancers, with other HDACi currently in clinical trials. The approved hydroxamic acid HDACi drugs chelate Zn 2+ in the eleven zinc-dependent HDAC enzymes, and despite being potent, HDACi generally exhibits limited selectivity between isoforms. 5 The disulfide prodrug romidepsin lacks selectivity between HDAC1, 2, 3, 10, and 11. 5 A lack of HDAC isoform selectivity among approved HDAC drugs might contribute to the undesired side effects associated with these drugs. 6−8 Additionally, it has also been proposed that the rearrangement of the hydroxamic acid functional group present in many HDACi drugs to an isocyanate can lead to mutagenicity. 9 Toward more efficacious HDAC therapeutics with reduced side effects, a number of studies have demonstrated that the selective targeting of HDAC1/2 and/or HDAC3 may be advantageous for specific diseases. 10−15 For example, selective inhibitors of HDAC1/2 were more effective at inducing apoptosis in B-cell acute lymphoblastic leukemia compared to other B-cell malignances, 10 while cutaneous T-cell lymphoma cell lines exhibited enhanced sensitivity to an HDAC3 selective inhibitor. 13 Additionally, each of the individual corepressor complexes that incorporate HDAC1/2 and 3 has a distinct cellular function, and therefore, the selective targeting of individual complexes may have potential therapeutic benefits in differing clinical applications. 16,17 Investigating novel approaches to target HDAC1/2 and 3, we previously reported benzamide-based Von Hippel−Lindau (VHL) E3-ligase proteolysis targeting chimeras (PROTACs) as an alternative strategy to degrade, rather than inhibit, enzyme activity. 18 PROTACs consist of a ligand for the protein of interest (POI), an E3-ligase ligand, and a linker that covalently bonds the two ligands. 19 PROTACs recruit the endogenous ubiquitination machinery via the E3-ligase to polyubiquitinate the POI, tagging it for degradation by the proteasome. 20,21 PROTAC 1 (JPS004) was based on the benzamide inhibitor CI-994, which exhibits selectivity for HDAC1/2 and 3 ( Figure 1). 18 We discovered a dependence on the linker length for HDAC1/2 and 3 degradation. Alkyl linkers consisting of 12 carbon atoms resulted in HDAC1/2 and 3 degradation in HCT116 colon cancer cells, while alkyl linkers of 6 carbon atoms, although inhibiting the HDAC1/ CoREST complex in vitro, showed no activity in cells. The VHL E3-ligase ligand, 21 in combination with the 12-atom alkyl linker, resulted in the most effective degradation. We wanted to carry out optimization studies of 1 with the aim of discovering novel PROTACs with enhanced degradation of HDAC1/2 and 3 and with differing selectivity profiles between these enzymes, allowing us to study the effects of removing these enzymes from the cell via proteasome-mediated degradation. To achieve this, we synthesized 23 novel heterobifunctional molecules making rationalized modifications to the benzamide, linker, and VHL E3-ligase ligand components (Chart 1). As HDAC1/2 and 3 also play an important role in the chromatin structure and transcription, for the first time, we also wanted to test the ability of such PROTACs to regulate global gene expression.

■ RESULTS
We first wanted to investigate the effects of the PROTAC linker length and composition on their ability to induce HDAC1/2 and 3 degradation. PROTACs were synthesized with alkyl linkers, alkyl linkers incorporating one or two oxygen atoms, poly ethylene glycol (PEG) linkers, and a piperazine substituted linker, with lengths ranging from 8 to 15 atoms ( Figure 2) (see the Experimental Section and the Supporting Information). Initially, each PROTAC was tested at 0.1, 1, and 10 μM in HCT116 cells for 24 h; then, cell extracts were prepared and evaluated for HDAC1/2 and 3 degradation by quantitative western blotting. For direct comparison, novel PROTACs were screened side by side with our original PROTAC 1 at a concentration of 10 μM, which previously caused maximum HDAC1/2 and 3 degradation ( Figure 2) (blots available in Supporting Information Figure S1). To Figure 2. (A) Compounds 2−13 were screened at 0.1, 1.0, and 10 μM with HDAC1, 2, and 3 abundance determined by quantitative western blotting with specific antibodies to HDAC1, 2, and 3 in HCT116 cells. CI-994 and 1 (JPS004) were also included at 10 μM. Error bars represent the standard deviation of two independent biological replicates. Statistical analysis of the significance of degradation for 1, 7, and 9 can be found in the Supporting Information ( Figure S7). (B) Representative western blots demonstrating degradation by 8 and 9 (JPS016). (C) H3K56ac blot and fold change at 10 μM. Error bars represent the average of two independent biological replicates.  Figure S2). There was a stepwise increase in HDAC1 degradation with increasing alkyl linker length from 9 to 11 carbon atoms (2, 3, and 4) at 1 and 10 μM (Figure 2A). The 11-atom linker, 4, exhibited HDAC1 degradation levels directly comparable to those of the 12-atom linker 1 at 10 μM. The same trend was also observed for HDAC2 with increasing alkyl linker length (2, 3, and 4); however, overall HDAC2 degradation was less pronounced in comparison to that of HDAC1. HDAC3 levels for 2, 3, and 4 were not greatly reduced with these subtle changes in linker length. H3K56ac levels also increased with increasing linker length (Figure 2Ccompare 2, 3, and 4), suggesting increased cell permeability and/or HDAC engagement with increasing linker length. The 11-atom linker 4 increased H3K56ac levels to the same degree as the 12-atom linker 1 ( Figure 2C). The 14-atom alkyl linker 5 exhibited comparable HDAC1 and HDAC2 degradation to the shorter linkers (1, 3, and 4); however, there was only a modest increase in H3K56ac compared to the shorter linkers, and we also noted solubility issues with 5.
The incorporation of one oxygen atom into 12-atom linkers 6 and 7 (JPS014) resulted in HDAC1 and HDAC2 degradation comparable to that of 1 and even enhanced for 7 at 10 μM, while HDAC3 degradation for both these PROTACs was also significantly enhanced compared to that of 1 surprisingly with the greater HDAC3 degradation at the lower concertation of 1 μM. Compounds 6 and 7 also increased H3K56ac to comparable or greater levels than CI-994 and 1.
The incorporation of two oxygen atoms into a 12-atom linker, 8, resulted in a loss of HDAC3 degradation compared to 6 and 7, while HDAC1 degradation was comparable to that of 1 but not maintained at 1 μM. However, H3K56ac levels for 8 matched those of CI-994, suggesting that this molecule, while not an effective degrader as other compounds in the library, can still act as a class I HDACi. Incorporating 2 oxygen atoms into a 15-atom linker, 9 (JPS016), resulted in enhanced degradation levels compared to those of 1 for both HDAC1 and HDAC3 even at 1 μM, while HDAC2 degradation was marginally increased compared to 1 at 10 μM (Figure 2A,B). This degradation was mirrored with increased H3K56ac levels to the same levels as CI-994 ( Figure 2C).
The compounds that incorporated PEG linkers, 10, 11, and 12, or a piperazine, 13, resulted in an almost complete loss of HDAC1/2 degradation; HDAC3 degradation was also generally compromised. Additionally, compounds 10, 11, 12, and 13 did not increase H3K56ac levels compared to the dimethyl sulfoxide (DMSO) control, suggesting that in HCT116 cells, these compounds do not act as degraders or inhibitors; we speculate that these compounds may not be reaching their class I HDAC targets in the nucleus. Overall, PROTACs 7 and 9 enhanced degradation compared to 1, with 9 showing enhanced degradation for HDAC1 and HDAC3 at 1 μM.
We next sought to investigate substitutions on the benzamide HDAC ligand of the PROTAC as it has been previously reported that substitutions with a fluorine atom on the 4-position of the anilide can increase selectivity for HDAC3, 23,24 while the introduction of a thiophene heterocycle on the 5-position of the anilide can enhance HDAC1/2 inhibitory potency and selectivity. 25 The 12-carbon linker with a fluorine atom, 15, directly analogous to 1, exhibited enhanced HDAC3 degradation compared to 1; however, despite this increase, HDAC1 degradation was still marginally elevated over HDAC3 at 10 μM ( Figure 3). For the 15-atom linker, 17, HDAC3 degradation was also enhanced at 1 μM compared to 1 and degradation levels for HDAC3 were now greater than those of HDAC1 and HDAC2; however, significant HDAC1 degradation was also still observed at 1 μM. The remaining fluorine-functionalized molecules 14 and 16 exhibited no gains in HDAC3 selectivity, with the PEG linker 16 exhibiting only modest HDAC3 degradation at 10 μM. Compounds 14−17 did not increase H3K56ac to the same levels as 1 or CI-994, with only 17 exhibiting a greater than twofold increase in H3K56ac compared to the DMSO control (see Supporting Information Figure S2).
Introduction of the thiophene moiety unfortunately did not result in enhanced degradation potency of HDAC1/2 in 18, 19, or 20. However, 20, with the PEG linker, did increase H3K56ac to levels similar to 1, suggesting that this molecule can act as an inhibitor ( Figure S2). Apart from the PEG linker analogue, 20, we also noted that the thiophene-substituted analogues exhibited exceptionally poor aqueous solubility. Overall, aside from a modest enhancement in HDAC3 degradation levels comparatively to HDAC1 and HDAC2 with 17, substitutions on the benzamide did not influence degradation selectivity or potency greatly. This perhaps suggests that formation of the ternary complex between the HDAC and VHL E3-ligase is more important for degradation than the affinity of the HDAC ligand in the PROTAC, which has been reported in other PROTACs also utilizing lower affinity ligands for the POI. 26,27 We wanted to investigate modifying the VHL E3-ligand as it had been previously shown that modifying the VHL-E3 ligand connectivity to the linker can modify the degradation selectivity profile of the PROTAC overall ( Figure 4). 26 At 10 μM, 21 (JPS035) exhibited comparable HDAC1 and HDAC2 degradation to 1, while 22 (JPS036) exhibited a reduction in HDAC1 and HDAC2 degradation compared to 1. However, in addition, the fluorinated cyclopropane VHL analogue, 22, reported to have higher affinity for VHL-E3 ligase than the acetyl VHL analogue in 21, 28 exhibited significantly enhanced HDAC3 degradation compared to 1 at both 1 and 10 μM. This may suggest that recruitment of the VHL E3-ligase with 22 is more favorable toward forming a ternary complex with HDAC3 over HDAC1/2. Compound 21 increased H3K56ac levels significantly but not to the same levels as 1, while the more prominent HDAC3 degrader 22 did not alter H3K56ac levels ( Figure S2). Analogues 23 and 24 exhibited only modest degradation of HDAC1, and these compounds did not increase H3K56ac levels greater than the DMSO control ( Figure S2).
Physiochemical property predictions of 1−24 were calculated using SwissADME 29 and compared with the maximal degradation values observed for HDAC1, HDAC2, and HDAC3 with 1−24 (Table S1). The majority of molecules that exhibited ≥50% maximal degradation of either HDAC1, HDAC2, or HDAC3 had a clogP of ≥ 5.0 and topological polar surface area (TPSA) values of ≤ 242.6 Å 2 with 8 and 23 being the only exceptions. The remaining molecules (exhibiting less than 50% maximal degradation of HDAC1, HDAC2, or HDAC3) had clogP values of < 5.0 with four exceptions, three of these exceptions exhibiting TPSA values > 242.6 Å 2 . Overall, in designing future class I HDAC PROTACs, in terms of physiochemical properties, maintaining a clogP of ≥ 5.0 and TPSA of ≤ 242.6 Å 2 may serve as potential guidelines.
We next sought to determine DC 50 values for PROTACs 7, 9, and 22, which all exhibited >50% degradation for HDAC1 and/or HDAC3 at 1 μM, while 21 was also chosen for direct comparison to structurally similar 22. 7 and 9 maintained submicromolar DC 50 values for HDAC1 and HDAC3, with 7 displaying DC 50 values of 0.91 ± 0.02 and 0.64 ± 0.03 μM for HDAC1 and HDAC,3 respectively ( Figure 5); 9 exhibited near-identical DC 50 values of 0.55 ± 0.18 and 0.53 ± 0.13 μM for HDAC1 and HDAC3, respectively. However, there was a notable observation in the dose−response curves of 7 and 9 for HDAC3 (all containing an amide bond to the L-tert-leucine residue of VHL); these PROTACs did not exhibit a standard dose−response curve for HDAC3 ( Figure 5). At concentrations greater than 1 μM, HDAC3 abundance increased rather than decreased, similar to the trend observed in the initial screening ( Figure 2). This looks like a hook effect for HDAC3, while at concentrations greater than 1 μM, HDAC1/ 2 levels continue to decrease, suggesting that at higher concentrations, HDAC3 degradation is compromised over HDAC1/2 degradation for these PROTACs. Intriguingly, this hook effect on HDAC3 was lost with PROTACs 21 and 22 (all containing an ether bond to the substituted phenyl substituent of VHL), with 22 exhibiting much more selective HDAC3 degradation over HDAC1/2. 21 and 22 now exhibited greater maximal degradation for HDAC3 over HDAC1, in comparison to 7 and 9, which exhibit greater maximal degradation for HDAC1 over HDAC3. Notably, 22 exhibited a DC 50 value of 0.44 ± 0.03 μM for HDAC3 and a Dmax value of 77% for HDAC3, with the least HDAC1 and HDAC2 degradation (Dmax values 41 and 18%, respectively) compared to the other three PROTACs. One explanation for the loss of the hook effect and enhanced selectivity for HDAC3 in 21 and 22 could be the differential orientation of the recruited VHL E3-ligase in ternary complex formation compared to 7 and 9. 26 We also determined the IC 50 values for 7, 9, 21, and 22 and CI-994 with the purified HDAC1-LSD1-CoREST complex, HDAC2-LSD1-CoREST complex, and HDAC3-SMRT complex ( Figures 5 and S8). CI-994 exhibited IC 50 values of 0.53 ± 0.09 μM for HDAC1 and 0.62 ± 0.07 μM for HDAC2 in the CoREST complex and 0.13 ± 0.01 for the HDAC3-SMRT complex, comparable to the previous literature. 30 The IC 50 values for 7 and 9 remained in the submicromolar range for all   Figure S4) and 10 μM ( Figure 6A,B). Notable HDAC1/2 degradation was observed after only 4 h at 10 μM, and degradation continued to increase over the 48 h time period, reaching Dmax values of 84% for HDAC1 and 51% for HDAC2 ( Figure 6A). A twofold increase in H3K56ac was observed compared to the DMSO control after 8 h, reaching a maximum fold change after 36 h ( Figure 6B). At 1 μM, a similar trend was observed for HDAC1/2 degradation ( Figure S4); however, maximal degradation was achieved after 24 h, and at 36 and 48 h, HDAC1/2 levels increased, possibly indicating inactivation of 9 at 1 μM by metabolism or other pathways. At 10 μM, over 24 h, little degradation was observed for HDAC3, as previously seen due to the hook effect ( Figure  6A). However, at 36 and 48 h, HDAC3 degradation reached approximately 50%; we speculate that the metabolism of 9 may reduce its concentration, whereby the hook effect is negated for HDAC3. At 1 μM, HDAC3 degradation was apparent from 4 h, and degradation reached maximum levels after 15 h ( Figure S4). This was followed by HDAC3 levels starting to increase after 24 h, again supporting a possible time-dependent inactivation of 9 at 1 μM.
To confirm that degradation was occurring via the proteasome and VHL E3-ligase, we synthesized a modified compound of 9 with the inactive VHL diasteroisomer, which as expected compromised degradation (see the Supporting Information, compound 25 and Figure S5). We also performed control experiments to investigate the effects on degradation in the presence of the proteasome inhibitor MG132 and competition experiments with the VHL ligand itself ( Figure  S5). The proteasome inhibitor alone modestly affected HDAC3 levels; however, despite this, degradation was still compromised in all other control experiments, providing strong evidence that 9 is recruiting the VHL E3-ligase to degrade HDAC1, 2, and 3 via the proteasome.
As class I HDACs exist in multiprotein corepressor complexes in vivo, we next wanted to determine the effects of PROTACs on components of these complexes. 1 HDAC1/2 and 3 all contribute structurally to the integrity of their respective complexes; 31,32 we therefore hypothesized that loss of the HDAC following degradation should also effect the stability of their binding partners. PROTACs 1, 7, and 9 were screened for their effects on lysine-specific demethylase 1 (LSD1), a component of the CoREST complex, 33 and SIN3A central component of the SIN3 complex. After 24 h, modest reductions in LSD1 levels were observed for 9; however, after 48 h, when HDAC1/2 degradation is also more prominent, LSD1 was significantly reduced with all three PROTACs in comparison to the DMSO and CI-994 controls. The most potent HDAC1/2 degrader, 9, reduced LSD1 levels to approximately 40% of controls. After 24 h with 7 and 9, SIN3A levels were reduced to 50 and 60% abundance, However, we were surprised to observe that SIN3A levels were also significantly reduced in the presence of the inhibitor CI-994 especially after 48 h to near−same levels as PROTACs, suggesting that the effects are due to a combination of both HDAC inhibition and ubiquitin-dependent degradation pathways.
The effects of novel PROTACs on cell viability were investigated with 1, 7, 9, 21, and 22 using CellTiter-Glo and flow cytometry (Figure 7). After 24 h, 7 and 9, the more potent HDAC1/2 degraders, had the highest percentage of cells in the sub-G1 phase, indicating substantial cell death when treated with PROTACs. After 48 h, 9 had the highest percentage of cells in the sub-G1 phase, followed equally by 1, 7, and Cl-994. A similar trend was observed in the CellTiter-Glo assay after 48 h with cells showing the greatest sensitivity to treatment with 9, 1, and 7 exhibiting EC 50 values of 5.2 ± 0.6, 4.3 ± 0.5, and 7.3 ± 0.5 μM, respectively, with the inhibitor CI-994 exhibiting an EC 50 value of 8.4 ± 0.8 μM. Interestingly, the HDAC3-selective PROTAC 22, DC 50 0.44 ± 0.03 μM and Dmax = 77% for HDAC3, and PROTAC 21 had little effects on cell viability (Figure 7). This implies that targeting HDAC1/2 is more important toward compromising cell viability in HCT116 cells than HDAC3. We also noted that at 10 μM, 21 exhibits effective HDAC1/2 and 3 degradation at the 24 h time point ( Figure 5) but does not compromise cell viability. However, in terms of DC 50, 21 is approximately a seven-and fourfold less-potent degrader of HDAC1 (20 DC 50 = 3.51 μM, HDAC1) than 9 and 7, respectively. We screened the inactive VHL diasteroisomer of 9 in flow cytometry with 9 and CI-994 ( Figure S9) to further probe the differences between inhibition and degradation. The population of cells in the sub-G1 phase was equal between 9 and the inactive VHL diasteroisomer of 9 after 24 and 48 h. This suggests that inhibition with 9 is as effective in compromising cell viability and likely reflects that 9 and presumably the inactive VHL diastereoisomer of 9 are also effective submicromolar inhibitors of HDAC1, HDAC2, and HDAC3 ( Figure 5). HDAC1/2 and 3 regulate global gene expression by manipulating histone acetylation levels across the genome. To examine the impact of PROTAC-mediated degradation on the HCT116 transcriptome, we performed RNA-seq with CI-994, 1, 7, 9, 21, and 22. Differential gene analysis ( Figure 8A) revealed substantial transcriptional changes resulting from the majority of the PROTACs used (p-adjusted value of < 0.01 and a log2 fold change of 1). PROTACs 1, 7, and 9 all displayed a striking phenotype, akin to CI-994. Differentially expressed gene (DEG) sets were subjected to gene ontology (GO) analysis. PROTAC treatment elicits a range of transcriptional changes to key cellular processes, including enrichment in cell cycle, apoptosis, and histone modification pathways ( Figure 8B). The pronounced change in cell cyclerelated genes is highlighted by the prominent downregulation of core regulatory factors, such as E2F1, CDK1, and cyclin E1, while there was upregulation of cell cycle inhibitors including p21 (CDKN1A) and p15 (CDKN2B) shown in Figure 8C. These changes are consistent between CI994, 1, 7, and 9, showing that both inhibition and degradation produce a strong antiproliferative phenotype in cancer cells. In addition, genes associated with apoptosis were also found to be significantly enriched ( Figure S10, see the Supporting Information), including proapoptotic TP63 and PMAIP1 and DHSRS2, which has been previously characterized in the promotion of HDACi-mediated apoptosis through the attenuation of MDM2-dependent p53 degradation. 34,35 There was a distinct correlation between the potency of degradation and the number of DEGs (compare Figure 5A with Figure 8D). PROTAC 9, identified as the most potent HDAC1/2 degrader and cytotoxic compound by flow cytometry, exhibited the greatest level of differential gene expression with 2464 and 1477 up-and downregulated DEGs, respectively. Compared to CI-994, both 7 and 9 appear to show an increased number of DEGs, consistent with their ability to promote apoptosis ( Figure 8D). In contrast, the less-potent HDAC1/2 and 3 degrader 21 showed approximately 10-fold less DEGs. However, perhaps even more interestingly, the HDAC3selective PROTAC 22 ( Figure 5A) showed the least effects on DEGs (Figures 8D and S10), indicating that HDAC1/2 compexes are the dominant HDAC isoforms in this cell type. Despite being relatively few, the majority of DEGs for 22 are upregulated, suggesting that HDAC3, as part of the NCoR/ SMRT complex, operates as a classical corepressor complex, while HDAC1/2-containing complexes play roles in both gene repression and active gene transcription.

■ DISCUSSION
Through modifications to the linker and VHL ligand of benzamide-based class I HDAC PROTACS, we have discovered 7 (JPS014), 9 (JPS016), and 22 (JPS036) submicromolar degraders of HDAC1 and/or HDAC3. Subtle alterations in the VHL ligand and attachment to the linker can have significant effects on the degradation profile of these PROTACs. For example, 7 and 9 exhibit the hook effect for HDAC3, while 21 and 22 exhibit a standard dose response curve for HDAC3. We unexpectedly found that substitution of the acetyl group for a fluorinated cyclopropane ring in VHL led to an HDAC3-selective degrader 22, although loss of HDAC3 alone did not cause significant cell death or changes in the transcriptome.
The more potent HDAC1/2 degraders 7 and 9 compromise LSD1 stability as part of the CoREST complex, highlighting a potential advantage to degrading class I HDACs, in addition to inhibition in situ. We also observed a strong correlation between HDAC1/2 degradation, induced cell death, and differential gene expression. PROTAC 9 appears to be improved in comparison to 1 with regards to changes in the transcriptome and apoptosis (Figures 7 and 8D). Both 7 and 9 showed a striking cell arrest phenotype when added to HCT116 cells with a significant reduction in proteins that promote G1/S transition, such E2F1, cyclin E1, and CDK2, While HDAC3 and HDAC6 degraders have previously been reported in the literature, 23,36−39 degraders of HDAC1 are less common. As far as we are aware, 7 and 9 are the first submicromolar degraders of HDAC1 reported. In a recent chemoproteomic study reported by Xiong et al. with PROTAC design based on pan-HDACi, HDAC3 and HDAC6 were found to be the most commonly degraded HDAC enzymes, with HDAC1/2 and HDAC9 being the least. 36 Complementary to this study, we have demonstrated through a focused VHL-recruiting benzamide PROTAC library that effective HDAC1/2 degraders can be obtained. We anticipate that there will be great interest in more potent HDAC1/2 degraders as potential therapeutics and as reagents to unlock the diverse function of different corepressor complexes. We are optimistic that a degradation strategy can be harnessed to target individual class I HDAC complexes selectively and thus generate improved therapeutics that retain the benefits of HDACi activity but with much reduced side effects.

■ CHEMISTRY
Heterobifunctional molecules 1−20 were prepared in five steps (Scheme 1). Monoprotected linkers (37a−e, 39a−c, 44a−b, 45, 47, and 51) were conjugated to substituted benzamides 35a−c by amide coupling with hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU). For the full synthesis and characterization of linkers and substituted benzamides, see the Supporting Information. The carboxyl protecting group in intermediates 52a−t was removed by base saponification or hydrogenolysis to yield intermediates 53a−t, which were conjugated to commercially available VH_032 amine via HATU-mediated amide coupling to give 54a−t. The Boc protecting groups in intermediates 54a−t were removed in trifluoroacetic acid (TFA)/dichloromethane (DCM), and after work-up, residual TFA was removed using a carbonate-based solid support resin, and final compounds 1−20 were purified by semipreparative high-performance liquid chromatography (HPLC) or column chromatography. Heterobifunctional molecules 21 and 22 were prepared in four steps (Scheme 2), the main difference to the preparation of 1−20 being a substitution reaction between 56 and VH_032 phenol in the preparation of 21 and 56 and VH_101 phenol in the preparation of 22. Compounds 23 and 24 were prepared by amide coupling via HATU with VH_032 phenol-alkylC4amine and 58 and 49b, respectively, followed by Boc removal.

■ EXPERIMENTAL SECTION
General Chemical Methods. All reagents were purchased from commercially available sources and used without further purification. VH_032 amine, VH_032 phenol, VH_101 phenol, and VH_032 phenol-alkylC4-amine were purchased from Tocris Bioscience. Preparative column chromatography and flash column chromatography using a Biotage Isolera purification system were both performed using silica gel 60 (230−400 mesh). Semipreparative HPLC was performed on a Thermo Fisher Ultimate 3000 system with Chromeleon software on a Phenomenex Luna C18 column. The mobile phases were water and acetonitrile with a flow rate of 10 mL/ min, 45 min gradient. NMR spectra were acquired using a Bruker 400 ( 1 H, 400 MHz; 13 C 101 MHz) instrument at ambient temperature using a deuterated solvent as a reference. High-resolution mass spectra (HRMS) were recorded on a Water Aquity XEVO Q ToF machine and measured in m/z. Analytical UPLC-MS data were collected on a Xevo G2-XS QToF mass spectrometer (Waters) coupled to an Acquity LC system (Waters) using an Acquity UPLC BEH C18 column (130 Å, 1.7 μm, 2.1 × 50 mm, Waters). The mobile and HATU (183.9 mg, 0.484 mmol) were added. The reaction mixture was stirred for 15 min, after which a solution of amine 35a (110.0 mg, 0.336 mmol) in DMF (2 mL) was added slowly, and the resultant solution was stirred at room temperature overnight. The reaction mixture was diluted in EtOAc (30 mL) and then washed with sat. NaHCO 3 (2 × 15 mL) and sat. NaCl (2 × 15 mL). The organic layer was dried over MgSO 4 , filtered, and concentrated in vacuo to give the corresponding crude, which was chromatographically purified (0−100% EtOAc in hexane) to afford 52g (157.4 mg, 0.247 mmol, 73% yield) as a colorless tar.
To a solution of the benzyl ester-protected HDACi-linker conjugate 52g (120.2 mg, 0.190 mmol) in tetrahydrofuran (THF), Pd/C (10% wt) was added. The reaction flask was filled with nitrogen and evacuated three times using a Schlenk line, before a balloon of hydrogen was added, and the resultant mixture was stirred vigorously overnight. The reaction mixture was filtered through a glass microfiber filter paper, and the filtrate was concentrated in vacuo to afford 53g (105.6 mg, 0.189 mmol, 99% yield) as a white solid.